The National Center for Microscopy and Imaging Research (NCMIR) was established to develop computer-aided advanced microscopy for acquisition of structural and functional data in the dimensional range of 1 nm to 100 um, a range encompassing macromolecules, subcellular structures and cells. With novel specimen staining methods, imaging instruments and computational capabilities, researchers are addressing the next great biological challenges in the post-genomic age by situating proteins and macromolecular complexes in their cellular and tissue environments. Resource instruments include intermediate high voltage transmission electron microscopes (IVEMs) and high-speed large-format laser-scanning light microscopes specially modified for "mesoscale" biological microscopy. Our collaboration, service, training and dissemination programs expand the development and use of these technologies to maximize their value to the biomedical community. In this application, NCMIR continues to develop novel methods for imaging biological systems across scales, building upon our successes during the previous funding period and expanding into new areas of technology development. Core technology projects are proposed to develop new staining methods, imaging modes and computational tools for correlated 3D imaging using unique optical and higher voltage electron microscopes. Core research is driven by collaboration with leading scientists in the US and around the world who are addressing key questions in basic cell and neurobiology and conducting translational research in neurodegenerative disease, stroke, cancer, heart failure and infectious disease, to name a few. A unique focus of this resource continues to be the utilization of advanced computational infrastructure to allow remote access to instruments, data and computational resources and to foster collaborations among widely distributed scientists. These new technologies were substantially enhanced over the preceding funding period and will be deployed within our resource to support core and collaborative research. Through this infrastructure, we provide researchers greater access to the Resource through web-based remote control systems for the optical and higher voltage electron microscopes, processing workflows for wide field fluorescence microscopy and electron tomography, and interfaces for computational grids and distributed databases via high-speed networks. In this proposal, we expand these activities to include web-based informatics tools for managing, searching, mining and sharing large multidimensional data sets. These data are shared with the public through the Cell Centered Database, a database for 2D, 3D and 4D microscopy data.